J. Med. Chem. 2000, 43, 2915-2921
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Synthesis and Biological Evaluation of Certain r,β-Unsaturated Ketones and Their Corresponding Fused Pyridines as Antiviral and Cytotoxic Agents† Hussein I. El-Subbagh,*,‡ Suhair M. Abu-Zaid,‡ Mona A. Mahran,‡ Farid A. Badria,# and Abdulrahman M. Al-Obaid‡ Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh-11451, Saudi Arabia, and Department of Pharmacognosy, College of Pharmacy, Mansoura University, Mansoura 35516, Egypt Received January 28, 2000
A new series of 3,5-bis(arylidene)-4-piperidones, as chalcone analogues carrying variety of aryl and heteroaryl groups, pyrazolo[4,3-c]pyridines, pyrido[4,3-d]pyrimidines, and pyrido[3,2-c]pyridines, carrying an arylidene moiety, and a series of pyrano[3,2-c]pyridines, as flavone and coumarin isosteres, were synthesized and screened for their in vitro antiviral and antitumor activities at the National Cancer Institute (NCI). Compounds 9 and 18 proved to be active against herpes simplex virus-1 (HSV-1), while compound 13 showed moderate activity against human immunodeficiency virus-1 (HIV-1). Compounds 14, 26, 28, 33, and 35 exhibited a broad spectrum antitumor activity. In addition, compounds 26, 33, and 35 proved to be of moderate selectivity toward leukemia cell lines. The pyrano[3,2-c]pyridines heterocyclic system proved to be the most active antitumors among the investigated heterocycles. Introduction
Chart 1
The cytotoxic activity of (E)-3,5-bis(benzylidene)-4piperidones 1 (Chart 1) and their specificity toward leukemia cell lines with IC50 values less than 10 µM have been reported.1 This type of compound is a combination of cyclic R,β-unsaturated ketone (chalcone) and β-amino ketone in one structure. Those compounds proved to possess marked affinities for thiols but not for amino or hydroxy functions found in nucleic acids; hence mutagenic and carcinogenic side effects should be absent.2,3 The practice of incorporating chalcones into heterocyclic nitrogenous ring has been noticed recently4 represented by the potential antiviral compounds Sglucosylated hydantoins 2 (Chart 1). This combination rationale has been used in our laboratory5,6 to synthesize some 5-substituted-2-thiohydantoin analogues as a novel class of antitumor agents. This latter study showed that compounds such as 3 and its S-glucosylated analogue 4 (Chart 1) are potential broad-spectrum antitumor agents6 based on the anticancer screening data obtained from the National Cancer Institute (NCI) antitumor drug discovery screen. The present study is a continuation of our previous efforts5,6 aiming to locate novel synthetic lead compound(s) for future development as antiviral and antitumor agents. A new series of (E)-3,5-bis(arylidene)-4piperidones (8-14) were designed carrying a variety of aryl and heteroaryl groups to explore the scope and limitations of their antiviral and antitumor activities. To determine the influence of the pharmacophoric function (R,β-unsaturated ketone) on activity, a selective † Presented in part at the 5th International Saudi Pharmaceutical Conference, Oct 26-28, 1999, Riyadh, Saudi Arabia. * Corresponding author. Phone: 966-1-467-7366. Fax: 966-1-4676383. E-mail:
[email protected]. ‡ King Saud University. # Mansoura University.
reduction of the unsaturated centers of 8-14 produced either the saturated ketone analogues 15-17 or the unsaturated alcohols 18-20. This type of tailored synthesis is aiming to discover the vital part of the pharmacophore, whether the ketonic moiety or the olefinic function. The finding that some 2-aminoquinazolines 5, their aza analogues 67 (Chart 1), and the other fused pyridine analogues such as 5-deazaaminopetrin8 and quinolones9 interfere with folic acid synthesis rationalized the design and synthesis of the pyrazolo[4,3-c]pyridine (21-26), pyrido[4,3-d]pyrimidine (27-29), and pyrido[3,2-c]pyridine (30-32) targets carrying an arylidene moiety to be evaluated as antitumor agents which may exert their
10.1021/jm000038m CCC: $19.00 © 2000 American Chemical Society Published on Web 07/07/2000
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Journal of Medicinal Chemistry, 2000, Vol. 43, No. 15
Scheme 1
El-Subbagh et al.
Scheme 2
Scheme 3
activity through folic acid synthesis inhibition. Several reports stated the ability of flavonoids to reduce the rate of colon,10-20 breast,21,22 and ovarian23 cancers. Flavonoids24 and coumarins25,26 are also proved to be HIVintegrase inhibitors. These literature facts suggested the synthesis of their isosteric analogues, the pyrano[3,2c]pyridines 33-35. The main objective of the present investigation, aiming toward the synthesis of the target compounds 8-35, is to reach a new active antiviral and antitumor agent(s) with potential activity toward cancerous cells and less toxicity toward normal cells. Chemistry The designed target compounds depicted in Schemes 1-3 were obtained by reacting the starting material 1-methyl-4-piperidone (7) with variety of aromatic aldehydes under aldol condensation conditions to produce the R,β-unsaturated ketone analogues 8-14. The chalcone derivatives 8, 9, and 13 (R ) Ph, 4-ClPh, and 4-pyridyl, respectively) were previously reported.27,28 The chalcone analogues 11, 12, and 14 (R ) 4-CH3Ph, 4-CH3OPh, and 2-thienyl, respectively), representing aryl and heteroaryl examples, were selected to be used for further synthesis of the other target molecules. Compounds 11, 12, and 14 were subjected to catalytic hydrogenation using a Parr hydrogenator (Pd/C, H2(g), 50 psi) to reduce the olefinic function yielding the 3,5bis(arylmethyl) derivatives 15-17. The ketonic groups in the chalcones 11, 12, and 14 were exclusively reduced into their corresponding racemic alcohols 18-20, quantitatively using NaBH4 in methanol (Scheme 1). The R,βunsaturated derivatives 11, 12, and 14 were subjected to cycloaddition condensation reactions using methyl-
or phenylhydrazine to give their corresponding pyrazolo[4,3-c]pyridine analogues 21-26 (Scheme 2). The interaction of 11, 12, and 14 with thiourea in refluxing NaOBu in butanol gave the pyrido[4,3-d]pyrimidine derivatives 27-29 (Scheme 2). The chalcone function of 11, 12, and 14 was further utilized for another cyclocondensation reaction using cyanoacetamide in refluxing butanol containing catalytic amount of piperidine to afford the pyrido[3,2-c]pyridine derivatives 30-32 (Scheme 3). Reacting 11, 12, and 14 with malononitrile in refluxing butanol produced the pyrano[3,2-c]pyridine analogues 33-35 (Scheme 3).
R,β-Unsaturated Ketones and Their Fused Pyridines
Journal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2917
Table 1. In Vitro Anti-HSV-1 Testing Results compd 9 14 18 19 27 29 aphidicolind
%
reductiona 93 33 82 58 45 27 100
Table 2. In Vitro Anti-HIV-1 Testing Results IC50c
MACb µg/mL
µM
µg/mL
µM
0.02 0.1 0.02 0.02 0.1 0.1 0.005
0.07 3.3 0.06 0.06 0.27 0.29 0.02
0.2 0.2 0.2 0.04 0.07 0.15 0.2
0.69 0.66 0.63 0.12 0.19 0.58 0.59
a Percent (%) reduction in the number of viral plaques. b Minimum antiviral concentration (minimum molar concentration proved to be lethal to the viral population). c Cytotoxicity (compound concentration caused 50% loss of the monolayer present around the viral plaques). d Positive control.
Results and Discussion In Vitro Anti-Herpes Simplex-1 Virus (HSV-1). The antiviral testing was performed using Vero cells, HSV-1, and aphidicolin (0.005 µg/mL) as a positive control.29 Compound 9 showed the highest activity among the tested compounds and was able to reduce the number of viral plaques by 93%. Replacing the 4-chloro atom in 9 either by hydrogen atom as in 8 or by electron-donating functions (CH3, CH3O) as in 11 and 12 abolished the antiviral activity. Meanwhile, replacing the entire aromatic residue (benzylidene) by a heteroaromatic moiety such as thienylidene (14) decreased the antiviral potency to 33% reduction of the viral plaques. These results imply that the aryl part attached to the R,β-unsaturated ketone moiety is an essential pharmacophoric site. Saturation of the olefinic function of the R,β-unsaturated ketones 8-14 produced the arylmethyl analogues 15-17, which proved to be devoid of any antiviral activity. On the other hand, reduction of the ketonic function produced the racemic R,β-unsaturated alcohols 18-20 with reduced magnitude of activity and reversed concept of substitution. Compound 18 with an electron-donating substituent (R ) CH3Ph) showed 82% reduction in the HSV-1 plaques, and replacing the 4-methylphenyl function by a 4-methoxyphenyl as in 19 lowered the antiviral activity to 58% reduction of viral plaques. Cyclization of the chalcone analogues to produce the pyrido[4,3-d]pyrimidine-2-thione preserved the antiviral potency with a dramatic decrease in activity as shown in 27 (R ) 4-CH3Ph) and 29 (R ) 2-thienyl). Compounds 9, 14, and 18 showed cytotoxicity (IC50) to Vero cells equal to that of aphidicolin (0.2 µg/mL), while compounds 19, 27, and 29 proved to be cytotoxic (Table 1). In Vitro Anti-Human Immunodeficiency-1 Virus (HIV-1). The procedure used to evaluate the anti-HIV-1 potency is designed to detect agents acting at any stage of the virus reproductive cycle.30 Compound 13 proved to possess moderate activity against HIV-1 cytopathic effect. Compounds 23-25, 30, and 32 did not show any activity or toxicity at the highest concentration used (200.0 µM), probably because of a solubility problem in the used culture media. Compounds 14, 20, 26-29, 31, and 33-35, however, were highly cytotoxic to the uninfected T4 cells at the concentrations which showed marginal antiviral activity in the infected cultures (Table 2). Antitumor Screening. Compounds 14, 20, 23-31, and 33-35 were subjected to the NCI’s in vitro disease-
compd
IC50a (µM)
EC50b (µM)
TI50c (IC50/EC50)
13 14 20 23 24 25 26 27 28 29 30 31 32 33 34 35 AZTe
>200.0 15.2 126.0 >200.0 >200.0 >200.0 9.1 27.6 6.8 34.0 >200.0 104.0 >200.0 11.1 30.4 9.0 35.6
64.7 d d d d d d d d d d d d d d d 0.0007
>3.09 d d d d d d d d d d d d d d d 50.59
a 50% Inhibitory concentration (molar concentration of compounds which cause 50% inhibition of cell growth). b 50% Effective concentration (molar concentration of compounds which cause 50% protection against HIV cytopathic effects). c Therapeutic index (the ratio of 50% inhibitory concentration to 50% effective concentration for each of the active compounds). d Inactive compounds. e Positive control.
oriented human cells screening panel assay.31,32 Three response parameters, median growth inhibition (GI50), total growth inhibition (TGI), and median lethal concentration (LC50), were calculated for each cell line. The NCI antitumor drug discovery screen has been designed to distinguish between broad-spectrum antitumor and tumor or subpanel-selective compounds.33 In the present study, the tested analogues showed a distinctive potential pattern of selectivity as well as broad-spectrum antitumor activity. With regard to sensitivity against individual cell lines, compounds 26, 28, 29, 34, and 35 showed effectiveness toward HL-60 (TB) leukemia cell line at a concentration range of 0.82.2 µM. Non-small-cell lung HOP-92 cell line proved to be sensitive toward compounds 23, 27, 33, and 35 at a concentration range of 0.08-2.0 µM. Compound 35 proved to be effective against renal ACHN, breast MCF7, and T-47D cell lines at concentrations of 0.3, 0.2, and 0.9 µM, respectively. With regard to broad-spectrum antitumor activity, compounds 14, 26, 28, and 33-35 showed GI50, TGI, and LC50 (MG-MID) < 100 µM against leukemia, nonsmall-cell lung, colon, CNS, melanoma, ovarian, renal, prostate, and breast cancer subpanel cell lines. Compounds 20, 23, 27, 29, and 31 showed (MG-MID) values < 100 µM at only the GI50 and TGI levels. Compounds 24 and 25 are the least effective members of this series with GI50 (MG-MID) values, while compound 30 proved to be inactive (Tables 3, 4). The ratio obtained by dividing the compounds full panel MG-MID concentration (µM) by its individual subpanel MG-MID concentration (µM) is considered as a measure for the compound selectivity.33 Ratios between 3 and 6 refer to moderate selectivity, while ratios greater than 6 indicate selectivity toward the corresponding cell line subpanel. Compounds 26 and 33-35 showed moderate selectivity at the GI50 level toward leukemia cell lines (Table 3). Compounds 33 and 35, beside their selectivity at the GI50 level, showed the same effectiveness at the TGI level. The in vitro antitumor evaluation of the newly synthesized com-
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El-Subbagh et al.
Table 3. Median Growth Inhibitory Concentration (GI50, µM) of in Vitro Subpanel Tumor Cell Lines subpanel tumor cell linesb compda
I
II
III
IV
V
VI
VII
VIII
IX
MG-MIDc
14
10.6 (2.2)d 32.5 (2.4) 31.8 (2.1) 37.4 (2.2) e (0.7) 2.2 (3.9) 19.2 (1.8) 2.0 (2.5) 22.4 (2.4) 35.0 (1.5) 5.1 (3.3) 9.1 (3.7) 3.2 (3.6)
36.5
23.7
36.6
34.6
23.5
24.2
23.9
28.7
23.5
83.9
85.8
e
92.5
81.4
96.2
e
84.3
77.1
86.4
79.2
80.7
85.0
74.4
89.7
59.5
86.0
68.0
97.4
e
77.0
77.9
92.8
83.7
69.5
e
81.0
82.1
e
63.1
77.7
80.7
54.7
e
91.8
72.9
20.8
3.8
35.7
42.5
24.2
65.3
19.3
23.6
8.5
59.6
37.1
53.7
58.8
51.2
60.8
38.3
39.4
34.5
6.5
4.0
30.0
11.7
5.0
5.9
9.1
11.1
4.9
67.2
70.5
76.1
60.5
65.5
75.0
e
45.1
53.5
66.6
60.5
44.9
79.9
51.8
61.2
70.9
66.9
52.5
(3.3)
16.6
37.7
26.6
15.5
18.1
29.4
30.7
16.9
36.7
41.2
60.5
70.7
38.1
40.4
79.4
48.6
33.3
16.3
17.3
42.2
36.3
25.0
32.5
13.5
27.9
11.6
20 23 24 25 26 27 28 29 31 33 34 35
a Compound 30 showed GI values > 100 µM. b I, leukemia; II, non-small-cell lung cancer; III, colon cancer; IV, CNS cancer; V, melanoma; 50 VI, ovarian cancer, VII, renal cancer, VIII, prostate cancer; IX, breast cancer. c GI50 full panel mean-graph midpoint (µM). d GI50 selectivity ratios obtained by dividing the compound’s full panel MG-MID (µM) by its leukemia subpanel MG-MID (µM) are shown in parentheses. e Compound showed GI 50 values > 100 µM.
Table 4. Total Growth Inhibitory Concentration (TGI, µM) of in Vitro Subpanel Tumor Cell Lines subpanel tumor cell linesb compda
I
II
III
IV
V
VI
VII
VIII
IX
MG-MIDc
14
51.6
90.1
72.2
82.7
84.1
81.4
70.4
d
75.4
20 23 26
78.1 87.8 52.2
d d d
d d 87.1
d d d
d 97.7 d
d d d
d d d
d d d
96.8 d d
27 28
62.3 36.3
89.9 93.6
83.6 76.3
94.1 81.9
d 83.1
94.0 76.4
96.9 97.2
d 30.1
88.8 68.1
29 31 33
69.8 80.7 17.9
d d 54.1
d d 43.9
d d 83.6
d d 59.5
d d 55.7
d 87.8 54.5
d d 66.2
98.1 d 70.1
34
49.9
95.5
97.4
d
93.9
d
96.9
d
93.8
35
27.6
75.5
87.3
d
87.0
73.2
81.6
90.9
57.7
74.0 (97.7)e 97.7 97.7 72.9 (95.8) 86.5 56.3 (91.8) 95.8 95.8 51.1 (97.3) 85.6 (97.3) 57.8 (91.1)
a Compounds 24, 25, and 30 showed TGI values > 100 µM. b For subpanel tumor cell lines, see footnote b of Table 3. c TGI full panel mean-graph midpoint (µM). d Compound showed TGI values > 100 µM. e Median lethal concentrations (LC50, µM) are shown in parentheses.
pounds revealed the potential of this class of compounds as antitumor agents. The R,β-unsaturated ketone found in compounds 8-14 proved to be essential for antitumor activity. Compound 14 proved to be 3-fold more active than its racemic R,β-unsaturated alcohol counterpart 20. Cyclocondensation of 14 with methyl- or phenylhydrazines afforded the pyrazolo[4,3-c]pyridine analogues 21-26 at which the olefinic residue, remaining from the chalcone function, is kept at position 7 in addition to the introduced heterocylic ring. The 2-methyl derivative 23 exhibited one-third the potency of 14, while the 2-phenyl analogue 26 showed a remarkable increase in activity by almost 3-fold, compared with 14, in addition to a particular selectivity toward leukemia cell lines.
Cyclocondensation of the other two chalcones 11 (R ) CH3Ph) and 12 (R ) CH3OPh) with phenylhydrazine produced the 2-phenyl analogues 24 and 25 with almost a 10-fold decrease in activity compared with their 2-thienylidene derivative 26. Cyclocondensation of the mentioned chalcones with thiourea yielded the pyrido[4,3-d]pyrimidine analogues 27-29, which showed various antitumor potencies depending on the type of substituent. Compound 28 (R ) CH3OPh) is the most active member among the pyrimidine analogues. Replacing the methoxy function of 28 by a methyl group gave compound 27 with reduced activity. Changing the arylidene moiety of 27 and 28 by the heteroaromatic function 2-thienyl at position 4 and 2-thienylidene at position 8 produced compound 29
R,β-Unsaturated Ketones and Their Fused Pyridines
Journal of Medicinal Chemistry, 2000, Vol. 43, No. 15 2919
with 10-fold reduced activity. On the other hand, cyclizing the chalcone analogues with cyanoacetamide yielded the pyrido[3,2-c]pyridine analogues 30 and 31. Compound 30 (R ) CH3Ph) proved to be inactive toward the used tumor cell lines, and changing the methyl function by a methoxy group as in 31 moved the potency toward the active side with GI50 and TGI values. Reacting the chalcone analogues 11, 12, and 14 with malononitrile produced the pyrano[3,2-c]pyridines 3335. Compound 35 (R ) 2-thienyl) proved to be the most active member among this group followed by compounds 33 (R ) 4-CH3Ph) and 34 (R ) 4-CH3OPh). Compounds 33-35 proved to possess potential selectivity toward leukemia cell lines. Structure-Activity Correlation. The attempt to connect the variations in the backbone of the synthesized heterocycles and the obtained antiviral and antitumor data facilitated the deduction of the following general considerations: (1) The obtained screening results showed that the antiviral activity is embedded in only three out of the seven designed main structure formulas found in compounds 8-35: namely (E)-3,5-bis(arylidene)-4-piperidone, their (()-(E)-4-hydroxypiperidine analogues, and pyrido[4,3-d]pyrimidine-2-thione. (2) The pyrano[3,2-c]pyridine heterocyclic system proved to be the most active antitumor agent among the investigated heterocycles represented by compounds 33-35 followed by the pyrido[4,3-d]pyrimidines 27-29 and pyrazolo[4,3-c]pyridines 23-26, then the R,βunsaturated ketones represented by compound 14. (3) The pattern of substitution on those heterocyclic ring systems suggested that for the pyrazolo[4,3-c]pyridines, the introduction of a phenyl group at position 2 (24-26) favored the antitumor activity rather than a methyl function (23), while the introduction of a 2-thienylidene moiety at position 7 produced more active compounds > 4-(CH3O)benzylidene > 4-(CH3)benzylidene: 26 > 25 > 24, respectively. For the pyrido[4,3d]pyrimidine, the order of substitution at position 8 is 4-(CH3O)benzylidene > 4-(CH3)benzylidene > 2-thienylidene: 28 > 27 > 29, respectively, while for the pyrano[3,2-c]pyridine the order of substitution is 2-thienylidene > 4-(CH3)benzylidene > 4-(CH3O)benzylidene: 35 > 33 > 34, respectively.
Central Research Laboratory, College of Pharmacy, King Saud University. All of the new compounds were analyzed for C, H and N and agreed with the proposed structures within (0.4% of the theoretical values. 1H and 13C NMR spectra were recorded on a Varian XL 400 MHz FT spectrometer; chemical shifts are expressed in δ ppm with reference to TMS. Mass spectral data were obtained on a Shimadzu GC/MS QP 5000 apparatus. Thin-layer chromatography was performed on precoated (0.25-mm) silica gel plates; compounds were detected with a 254-nm UV lamp. Silica gel (60-230 mesh) was employed for routine column chromatography separations. The synthesis of compounds 8, 9, and 13 were previously reported in refs 27 and 28. The synthesized compounds were tested in vitro for their antiviral activity using HSV-1 at College of Pharmacy, University of Mansoura, Mansoura, Egypt. In vitro anti-human HIV-1 and antitumor activity were conducted at the NCI, Bethesda, MD. General Procedure for Preparation of (E)-1-Methyl3,5-bis(arylidene)-4-piperidones 10-12 and 14. A mixture of 1-methyl-4-piperidone (7; 0.01 mol) and the appropriate aldehyde (0.02 mol) in alcoholic NaOH (50 mL, 10%) was stirred at room temperature for 10 min. The separated solid was filtered and recrystallized from the suitable solvents. (E)-1-Methyl-3,5-bis(4-nitrobenzylidene)-4-piperidone (10). The crude product was recrystallized from BuOH to give 10 (65%): mp 204-5 °C; 1H NMR (CDCl3) δ 2.25 (s, 3H, NCH3), 2.85-3.56 (m, 4H, CH2NCH2), 6.55-8.89 (m, 10 H, CHdC & ArH); MS m/e (379, 20%). Anal. (C20H17N3O5) C, H, N. (E)-1-Methyl-3,5-bis(4-methylbenzylidene)-4-piperidone (11). The obtained crude product was recrystallized from EtOH/H2O to give 11 (95%): mp 195-6 °C; MS m/e (317, 100%). Anal. (C22H23NO) C, H, N. (E)-1-Methyl-3,5-bis(4-methoxybenzylidene)-4-piperidone (12). The crude product was recrystallized from EtOH to yield 12 (70%): mp 193-4 °C; MS m/e (349, 100%). Anal. (C22H23NO3) C, H, N. (E)-1-Methyl-3,5-bis(2-thienylidene)-4-piperidone (14). The obtained crude product was recrystallized from EtOH/H2O to produce 14 (84%): mp 114-5 °C; MS m/e (301, 100%). Anal. (C16H15NOS2) C, H, N. General Procedure for Preparation of 1-Methyl-3,5bis(arylmethyl)-4-piperidones 15-17. (E)-1-Methyl-3,5-bis(arylidene)-4-piperidone (11, 12, or 14; 0.01 mol) was dissolved in ethanol (50 mL) and subjected to catalytic hydrogenation (H2, 10% Pd/C, 50 psi) for 10 h. The reaction mixture was filtered through Celite and filtrate was evaporated to dryness in vacuo. The obtained residue was chromatographed on silica gel (20% EtOAc, CHCl3) to give an analytically pure samples of compounds 15-17, which were then recrystallized from the appropriate solvents. 1-Methyl-3,5-bis(4-methylphenylmethyl)-4-piperidone (15). The obtained product was recrystallized from EtOH/H2O to give 15 (32%): mp 167-8 °C; 1H NMR (CDCl3) δ 2.18 (s, 3H, NCH3), 2.34 (s, 6H, ArCH3), 2.85-3.56 (m, 10H, (CH2CHCH2)2N), 7.20-7.62 (m, 8H, ArH); MS m/e (321, 10%). Anal. (C22H27NO) C, H, N. 1-Methyl-3,5-bis(4-methoxyphenylmethyl)-4-piperidone (16). The obtained product was recrystallized from MeOH to yield 16 (20%): mp 200-2 °C; MS m/e (353, 15%). Anal. (C22H27NO3) C, H, N. 1-Methyl-3,5-bis(2-thienylmethyl)-4-piperidone (17). The obtained product was recrystallized from EtOH to yield 17 (15%): mp 156-7 °C; MS m/e (305, 12%). Anal. (C16H19NOS2) C, H, N. General Procedure for Preparation of (()-(E)-1-Methyl-3,5-bis(arylidene)-4-hydroxypiperidines 18-20. (E)-1Methyl-3,5-bis(arylidene)-4-piperidone (11, 12, or 14; 0.01 mol) was dissolved in methanol (50 mL) and stirred. NaBH4 (1.0 g) was added portionwise over a period of 0.5 h and stirred at room temperature for another 2 h. Solvent was removed under reduced pressure, 20 mL of water was then added, and the undissovled solid was filtered and recrystallized from the appropriate solvent.
Conclusion The R,β-unsaturated ketone 9 and the racemic alcohol 18 showed antiviral activity against HSV-1 and HIV-1, respectively. The heterocyclic systems pyrano[3,2-c]pyridine, pyrido[4,3-d]pyrimidine, and pyrazole[4,3-c]pyridine exhibited remarkable cytotoxic activity with selectivity toward leukemia cell lines possibly through interfering with folic acid synthesis. These heterocycles could be considered as useful templates for future development and further derivatization or modification to obtain more potent and selective antitumor agents. It is worth mentioning that compounds 14, 26, 28, 33, and 35 have been selected by the NCI biological evaluation committee for performing hollow fiber assay in vivo testing. Experimental Section Melting points were determined on a Mettler FP80 melting point apparatus and are uncorrected. Microanalyses were performed on a Perkin-Elmer 240 elemental analyzer at the
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El-Subbagh et al.
(()-(E)-1-Methyl-3,5-bis(4-methylbenzylidene)-4-hydroxypiperidine (18). The prepared compound was recrystallized from EtOH to yield 18 (96%): mp 129-30 °C; 1H NMR (CDCl3) δ 2.34 (s, 3H, NCH3), 2.37 (s, 6H, ArCH3), 3.31-3.76 (dd, 4H, J ) 15 Hz, CH2NCH2), 3.40 (s, 1H, OH), 4.66 (s, 1H, CHOH), 6.59 (s, 2H, CHdC), 7.16 (s, 8H, ArH); 13C NMR δ 21.7, 44.9, 56.0, 78.9, 125.9, 129.3, 129.5, 134.4, 137.1, 137.8; MS m/e (319, 100%). Anal. (C22H25NO) C, H, N. (()-(E)-1-Methyl-3,5-bis(4-methoxybenzylidene)-4-hydroxypiperidine (19). The synthesized compound was recrystallized from EtOH to give 19 in (90%): mp 138-9 °C; MS m/e (351, 65%). Anal. (C22H25NO3) C, H, N. (()-(E)-1-Methyl-3,5-bis(2-thienylidene)-4-hydroxypiperidine (20). The obtained compound was recrystallized from EtOH to produce 20 (87%): mp 141-2 °C; MS m/e (303, 67%). Anal. (C16H17NOS2) C, H, N. General Procedure for Preparation of (E)-2-(Methyl or phenyl)-3-aryl-5-methyl-7-arylidene-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridines 21-26. A mixture of 11, 12, or 14 (0.01 mol), the appropriate hydrazine derivative (0.04 mol) and Na metal (0.5 g) in ethanol (20 mL) was heated under reflux for 10 h. Solvent was then removed under reduced pressure and the remained residue was chromatographed on silica gel (CHCl3) to furnish compounds 21-26. (E)-2-Methyl-3-(4-methylphenyl)-5-methyl-7-(4-methylbenzylidene)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,d-c]pyridine (21). The prepared compound was recrystallized from EtOAc to produce 21 (28%): mp 118-20 °C; 1H NMR (CDCl3) δ 2.35 (s, 3H,NCH3), 2.37 (s, 3H, ArCH3), 2.42 (s, 3H, ArCH3), 2.79 (s, 3H, NCH3), 3.05-3.12 (m, 2H, N-CH2), 3.253.34 (m, 2H, CH2-N), 4.00 (d, 1H, J ) 11 Hz, CH-CH), 4.12 (d, 1H, J ) 14 Hz, CH-CH), 7.03-7.13 (m, 8H, ArH), 7.29 (s, 1H, CHdC); MS m/e (345, 100%). Anal. (C23H27N3) C, H, N. (E)-2-Methyl-3-(4-methoxyphenyl)-5-methyl-7-(4-methoxybenzylidene)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3c]pyridine (22). The produced compound was recrystallized from EtOAc to give 22 (35%): mp 182-3 °C; MS m/e (377, 85%). Anal. (C23H27N3O2) C, H, N. (E)-2-Methyl-3-(2-thienyl)-5-methyl-7-(2-thienylidene)3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine (23). The obtained product was recrystallized from EtOH to yield 23 (20%): mp 154-5 °C; MS m/e (329, 100%). Anal. (C17H19N3S2) C, H, N. (E)-2-Phenyl-3-(4-methylphenyl)-5-methyl-7-(4-methylbenzylidene)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine (24). The synthesized compound was recrystallized from EtOH to give 24 (53%): mp 175-6 °C; 1H NMR (DMSOd6) δ 2.36 (s, 3H, NCH3), 2.39 (s, 6H, Ar-CH3), 2.99-3.03 (dd, 2H, J ) 7.0, 6.5 Hz, NCH2), 3.66-3.75 (m, 2H, CH2N), 5.21 (d, 1H, J ) 5 Hz, CH-CH), 5.24 (d, 1H, J ) 10 Hz, CH-CH), 6.54-7.39 (m, 14H, CHdC & ArH); 13C NMR 21.5, 21.7, 42.8, 64.4, 113.9, 119.6, 121.3, 126.2, 126.9, 129.4, 129.9, 130.3, 133.0, 134.4, 137.6, 138.5, 139.9, 144.8, 149.0; MS m/e (407, 10%). Anal. (C28H29N3) C, H, N. (E)-2-Phenyl-3-(4-methoxyphenyl)-5-methyl-7-(4-methylbenzylidene)-3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3c]pyridine (25). The produced compound was recrystallized from CHCl3/EtOH to yield 25 (51%): mp 192-3 °C; MS m/e (439, 48%). Anal. (C28H29N3O2) C, H, N. (E)-2-Phenyl-3-(2-thienyl)-5-methyl-7-(2-thienylidene)3,3a,4,5,6,7-hexahydro-2H-pyrazolo[4,3-c]pyridine (26). The obtained product was recrystallized from CHCl3/Hexane to give 26 (36%): mp 169-70 °C; MS m/e (391, 100%). Anal. (C22H21N3S2) C, H, N. General Procedure for Preparation of (E)-4-Aryl-6methyl-8-arylidene-3,4,5,6,7,8-hexahydro-1H-pyrido[4,3d]pyrimidine-2-thiones 27-29. A mixture of the diarylidene compound 11, 12, or 14 (0.01 mol), thiourea (0.8 g, 0.01 mol), and Na metal (0.5 g) in butanol (50 mL) was heated under reflux for 10 h. Solvent was evaporated in vacuo, water (20 mL) was then added, and the mixture was neutralized to pH 6. The separated solid was filtered, washed, dried and recrystallized from the appropriate solvent.
(E)-4-Methylphenyl-6-methyl-8-(4-methylbenzylidene)3,4,5,6,7,8-hexahydro-1H-pyrido[4,3-d]pyrimidine-2thione (27). The obtained residue was recrystallized from EtOH/H2O to produce 27 (91%): mp 151-2 °C; 1H NMR (DMSO-d6) δ 2.37 (s, 3H, NCH3), 2.40 (s, 6H, ArCH3), 3.003.05 (dd, 2H, J ) 7.0 Hz, NCH2), 3.68-3.78 (m, 2H, CH2N), 5.28 (s, 1H, CHNH), 6.60-7.42 (m, 9H, CHdC & ArH), 8.04 (brs, 1H, NH), 8.39 (s, 1H, NH); MS m/e (375, 43%). Anal. (C23H25N3S) C, H, N. (E)-4-Methoxyphenyl-6-methyl-8-(4-methoxybenzylidene)-3,4,5,6,7,8-hexahydro-1H-pyrido[4,3-d]pyrimidine-2thione (28). The produced compound was recrystallized from EtOH/ether to give 28 (80%): mp 197-8 °C; MS m/e (407, 42%). Anal. (C23H25N3O2S) C, H, N. (E)-4-(2-Thienyl)-6-methyl-8-(2-thienylidene)-3,4,5,6,7,8hexahydro-1H-pyrido[4,3-d]pyrimidine-2-thione (29). The prepared compound was recrystallized from EtOH to produce 29 (83%): mp 217.8 °C; MS m/e (359, 100%). Anal. (C17H17N3S3) C, H, N. General Procedure for Preparation of (E)-3-Cyano-4aryl-6-methyl-8-arylidene-5,6,7,8-tetrahydro-1H-pyrido[3,2-c]pyridin-2-ones 30-32. A mixture of the diarylidene compound 11, 12, or 14 (0.01 mol) and cyanoacetamide (0.8 g, 0.01 mol) was refluxed in butanol (50 mL) in the presence of a catalytic amount of piperidine for 10 h. Solvent was removed under reduced pressure and the obtained residue was washed with water, dried and recrystallized from appropriate solvents. (E)-3-Cyano-4-(4-methylphenyl)-6-methyl-8-(4-methylbenzylidene)-5,6,7,8-tetrahydro-1H-pyrido[3,2-c]pyridin2-ones (30). The obtained compound was recrystallized from EtOH to give 30 (33%): mp 234-5 °C; 1H NMR (DMSO-d6) δ 2.17 (s, 3H, NCH3), 2.33 (s, 3H, ArCH3), 2.38 (s, 3H, ArCH3), 2.48-2.49 (m, 4H, CH2NCH2), 7.25-7.34 (m, 9H, CHdC & ArH), 7.71 (brs, 1H, NH); MS m/e (381, 53%). Anal. (C25H23N3O) C, H, N. (E)-3-Cyano-4-(4-methoxyphenyl)-6-methyl-8-(4-methoxybenzylidene)-5,6,7,8-tetrahydro-1H-pyrido[3,2-c]pyridin-2-ones (31). The synthesized compound was recrystallized from EtOH/H2O to give 31 (56%): mp 216-7 °C; MS m/e (413, 12%). Anal. (C25H23N3O3) C, H, N. (E)-3-Cyano-4-(2-thienyl)-6-methyl-8-(2-thienylidene)5,6,7,8-tetrahydro-1H-pyrido[3,2-c]pyridin-2-ones (32). The crude product was recrystallized from BuOH to give 32 (60%): mp 189-90 °C; MS m/e (365, 15%). Anal. (C19H15N3OS2), C, H, N. General Procedure for Preparation of (E)-2-Amino-3cyano-4-aryl-6-methyl-8-arylidene-5,6,7,8-tetrahydro-4Hpyrano[3,2-c]pyridines 33-35. A mixture of the diarylidene compound 11, 12, or 14 (0.01 mol) and malononitrile (0.7 g, 0.01 mol) in butanol (50 mL) was heated under reflux for 5 h. The precipitated product was filtered off and recrystallized. (E)-2-Amino-3-cyano-4-(4-methylphenyl)-6-methyl-8-(4methylbenzylidene)-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine (33). The crude product was recrystallized from EtOH to give 33 (72%): mp 214-5 °C; 1H NMR (DMSO-d6) δ 2.11 (s, 3H, NCH3), 2.27 (s, 3H, ArCH3), 2.29 (s, 3H, ArCH3), 2.48-2.97 (dd, 2H, J ) 18 Hz, NCH2), 3.22-3.47 (dd, 2H, J ) 15 Hz, CH2N), 3.97 (s, 1H, pyran-H), 6.77 (brs, 2H, NH2), 6.86 (s, 1H, CHdC), 7.09-7.20 (m, 8H, ArH); 13C NMR δ 21.0, 21.2, 44.9, 54.6, 54.9, 56.5, 113.3, 120.8, 121.7, 127.2, 127.9, 129.3, 129.5, 129.6, 133.5, 136.6, 136.9, 139.5, 140.9, 160.1; MS m/e (383, 59%). Anal. (C25H25N3O) C, H, N. (E)-2-Amino-3-cyano-4-(4-methoxyphenyl)-6-methyl-8(4-methoxybenzylidene)-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine (34). The resultant residue was recrystallized from EtOH to produce 34 (97%): mp 203-4 °C; MS m/e (415, 45%). Anal. (C25H25N3O3) C, H, N. (E)-2-Amino-3-cyano-4-(2-thienyl)-6-methyl-8-(2-thienylidene)-5,6,7,8-tetrahydro-4H-pyrano[3,2-c]pyridine (35). The crude product was recrystallized from EtOH/H2O to give 35 (65%): mp 190-2 °C; MS m/e (367, 100%). Anal. (C19H17N3OS2) C, H, N.
Acknowledgment. The financial support of King Abdulaziz City for Science and Technology, Grant AT-
R,β-Unsaturated Ketones and Their Fused Pyridines
17-T35, is acknowledged. Thanks are due to the NCI, Bethesda, MD, for performing the anti-HIV and antitumor testing of the synthesized compounds. The technical assistance of Mr. Tanvir A. Butt is greatly appreciated. References (1) Dimmock, J. R.; Arora, V. K.; Duffy, M. J.; Reid, R. S.; Allen, T. M.; Kao, G. Y. Evaluation of Some N-acyl analogues of 3,5-Bis (arylidene)-4-piperidones for Cytotoxic Activity. Drug Des. Discovery 1992, 8, 291-299. (2) Dimmock, J. R.; Raghavan, S. K.; Logan, B. M.; Bigam, G. E. Antileukemic Evaluation of Some Mannich Bases Derived from 2-Arylidene-1,3-diketones. Eur. J. Med. Chem. 1983, 18, 248254. (3) Baluja, G.; Municio, A. M.; Vega, S. Reactivity of Some R,βUnsaturated Ketones Towards Sulfhydryl Compounds and Their Antifungal Activity. Chem. Ind. 1964, 2053-2054. (4) El-Barbary, A. A.; Khodair, A. I.; Pederson, E. B.; Nielsen, C. s-Glucosylated Hydantoins as New Antiviral Agents. J. Med. Chem. 1994, 37, 73-77. (5) Khodair, A. I.; El-Subbagh, H. I.; El-Emam, A. A. Synthesis of Certain 5-Substituted-2-Thiohydantoin Derivatives as Potential Cytotoxic and Antiviral Agents. Bull. Chim. Farm. 1997, 136, 561-567. (6) Al-Obaid, A. M.; El-Subbagh, H. I.; Khodair, A. I.; El-mazar, M. M. A. 5-Substituted-2-thiohydantoin Analogues as a Novel Class of Antitumor Agents. Anticancer Drugs 1996, 7, 873-880. (7) Lorand, T.; Deli, J.; Szabo, D.; Foldesi, A. Potentially Bioactive Pyrimidine Derivatives. Pharmazie 1985, 40, 53-539. (8) Temple, C.; Elliot, R.; Montogomery, J. Pyrido[2,3-d]pyrimidines. Synthesis of the 5-Deaza Analogues of Aminopterin, Methotrexate, Folic Acid. J. Org. Chem. 1982, 47, 761-764. (9) Li, L.; Wang, H.; Kuo, S.; Wu, T.; Mauger, A.; Lin, C. Antitumor Agents 155. Synthesis and Biological Evaluation of Substituted 2-Phenyl-4-Quinolones. J. Med. Chem. 1994, 37, 3400-3407. (10) Ambika, D. M.; Das, N. P. In Vitro Effects of Natural Plant Polyphenols on the Proliferation of Normal and Abnormal Human Lymphocytes and Their Secretion of Interleukin-2. Cancer Lett. 1993, 69, 191-196. (11) Cassady, J. M. Natural Products as a Source of Potential Cancer Chemopreventive Agents J. Nat. Prod. 1990, 53, 23-30. (12) Newmark, H. L. Plant Polyphenolics as Inhibitors of Mutational and Precarcinogenic Events. Can. J. Physiol. Pharmacol. 1986, 65, 461-466. (13) Ramanathan, R.; Tan, C. H.; Das, N. P. Cytotoxic Effects of Polyphenols and Fat Soluble Vitamins on Malignant Human Cultured Cells. Cancer Lett. 1992, 62, 217-224. (14) Narborne, J. B.; Maby, T. J. The Flavonoids: Advances in Research; Chapman and Hall: London, 1982. (15) Elliott, A. J.; Scheiber, S. A.; Thomas, C.; Pardini, R. S. Inhibition of Glutathione Reductase by Flavonoids: A Structure-activity Study. Biochem. Pharmacol. 1992, 44, 1603-1616. (16) Kuppusamy, U. R.; Das, N. P. Effect of Flavonoids on Cyclic AMP Phosphodiesterase and Lipid Mobilization in Rat Adipocytes. Biochem. Pharmacol. 1992, 44, 1307-1312. (17) Suolinna, E. M.; Buchskaum, R. N.; Racker, E. The Effect of Flavonoids on Aerobic Glycolysis and Growth of Tumor Cells. Cancer Res. 1975, 35, 1865-1870.
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